1. Field of the Invention
The present invention relates generally to cell lines which are generated from differentiated tissue of insects and which are susceptible to baculoviruses and may be used to replicate such viruses. The invention further relates to the use of such cell lines to replicate large numbers of baculoviruses and particularly, to replicate large numbers of genetically engineered baculoviruses, and to thus express large quantities of recombinant proteins. In addition, the invention relates to cell lines which tolerate the expression of a wide range of recombinant proteins including known toxins.
2. Description of the Relevant Art
Baculoviruses are considered a potentially important tool for managing insect pests and cell culture is a desired means of producing them. In nature, insects can become infected with baculovirus particles as a result of consuming food contaminated with baculovirus particles. These food-borne baculovirus particles are typically in the form of occlusion bodies (OB) which are composed of multiple viral particles embedded within a virus-encoded proteinaceous crystal. After ingestion, the protein crystal of the occlusion bodies dissolves, releasing individual virus particles which invade the epithelial cells that line the midgut. Viral replication takes place in the nuclei of the cells, and usually two forms of baculovirus, occluded and extracellular virus (ECV), are generated during replication. ECV is produced first and acquires an envelope as it buds out from the surface of the cell. The ECV can then infect other cells within the insect, including fat body cells, epidermal cells, and hemocytes. Following this initial stage of infection, virions are produced which are occluded in OB. OB formation continues until the cell ultimately dies or lyses. Some baculoviruses can infect virtually every tissue in the host insect so that at the end of the infection process, the entire insect is liquified, releasing extremely large numbers of OB which are then responsible for spreading the infection to other insects (1986. The Biology of Baculoviruses, Vol. I and II. Granados et al., Eds. CRC Press, Boca Raton, Fla.).
The first attempts to replicate baculoviruses in cell culture were in lines from the homologous insect species (Grace, T. D. C. 1967. In Vitro 3: 104-117; Goodwin et al. 1970. J. Invertebr. Pathol. 16: 284-288). However, Sohi et a. (1972. J. lnvertebr. Pathol. 19: 51-61) and Granados et al. (1976. In: Invertebrate Tissue Culture, Applications in Medicine, Biology, and Agriculture, Kurstak et al., Eds. Academic Press, New York, N. Y. , pages 379-389) soon demonstrated the replication of baculoviruses in cell lines from insect species other than the ones from which the viruses were isolated. Quiot (1976. C. R. Acad. Sci. Ser. D. 282: 465-467) reported a gypsy moth cell line that replicated an iridovirus and seven baculoviruses, but not the gypsy moth nuclear polyhedrosis virus (NPV). Goodwin et aL (1978. In Vitro 14: 485-494) reported that several cell lines developed from gypsy moth by very similar methods were quite different in their replication of baculoviruses. Some cell lines replicated the gypsy moth NPV; whereas, others did not replicate gypsy moth NPV but did replicate the Autographa californica nuclear polyhedrosis virus (AcNPV).
AcNPV, in particular, has a broad in vivo host range and will multiply in cell lines from a number of species of insects. Some of the cell lines in which AcNPV has been replicated are shown in Table 1. In addition to 11 genera and 12 species, the listed cell lines are from a number of different tissues such as blood cells and minced whole larvae. The relative merits of this large variety of cell diversity has not been adequately screened to categorize its potential for virus production or gene expression.
The first comparison of AcNPV replication in cell lines from several species of insects was made by Lynn and Hink in 1980 (J. Invertebr. Pathol. 35: 234-240). Five cell lines known to be susceptible to AcNPV were evaluated using several parameters to measure performance. The cell line used in any in vitro system is an important element both in terms of the quality and the quantity of the final product. The selected cell line must be capable of growth in suspension in large volumes. The IZD-MB0503 line from Mamestra brassicae was the best of those tested based upon the yield of active polyhedra over several passages. However, cell lines from Trichoplusia ni Tn-368, produced polyhedra with specific activity closest to that of polyhedra produced in insect larvae. The line Sf-1254, from Spodoptera frugiperda, produced only about one-tenth the amount of ECV produced by most of the other cell lines. Thus, it would be difficult to use this line for production as either large volumes of culture supernatant would be required for seed virus or the virus would have to be concentrated.
The NPV of the gypsy moth is registered as a pesticide by EPA under the name GYPCHEK. Production of the virus for pest management is in insect larvae as the yields from cell lines developed by Goodwin (Goodwin et al. 1978, supra) have been too low and the process too costly. Therefore, cell lines for different gypsy moth tissues, embryonic and fat body, were developed by Lynn and screened for their production capabilities (Lynn et al. 1989. Appl Environ. Microbiol. 55: 1049-1051). A line from fat body, IPLB-LdFB, and one from embryos, IPLB-Elt, were compared with Goodwin""s line from pupal ovary, IPLB-Ld-652Y. OBs produced in the three lines are shown in FIG. 1. Three strains of the virus were used in the studies. The LdFB cells produced the highest number of OBs regardless of the virus strain used, with the LdFB-Ab combination producing significantly more than any other combination. ECV was not produced in high titers in any cell-virus combination. TCID50s/ml ranged from 3.83xc3x97103 to 2.61xc3x97105 in IPLB-652Y depending on the virus strain used. These results were obtained in attached cultures and based upon them, the LdFB-Ab virus combination was considered the best for in vitro production. However, attempts to scale up suspension cultures of the LdFB cells have not been successful as the cells are very fragile, especially after infection. Yields in suspension culture were considerably less than in static culture. Volkman et al. (1984. Appl. Environ. Microbiol. 44:227-233) made a comparison similar to the Lynn studies using an immunoassay to detect infected cells. All of the cell lines tested responded linearly to virus dose; however, the dose range for Tn-368 cells, the most sensitive, was 4.5-5.5 log10 and the range for the least sensitive cells, from L. dispar, was 1.3-2.3 log10. Cell lines from Bombyx mori and L. dispar produced more than 99% single cell foci of infection. One possible interpretation of these results is that these cell lines do not produce much ECV, making them less suitable for production.
In the most extensive screening of variation in virus susceptibility, Miltenburger et al. (1984.Z. Naturforsch 39: 993-1002) challenged over 80 primary cultures derived from Cydia pomonella, apple codling moth, with Choristoneura murinana NPV and C. pomonella granulosis virus (GV). The primary cultures were obtained from embryonic tissues or tissues from fourth or fifth instar larvae. Several methods were employed for subculturing the cells to obtain a variety of cell populations in the new cultures. The majority of cultures supported some level of NPV replication, but only nine were judged to be very good. Of these nine, four were unstable, showing significant decline in the number of polyhedra produced within five passages. Seven supported at least minimal GV replication, but only two produced capsules as revealed by electron microscopy. One supported better than minimal, but not exceptional, GV replication.
In comparison with the AcNPV, the Helicoverpa zea NPV is host specific, infecting only H. zea and closely related species. McIntosh and colleagues (Lenz et al. 1991. J. Invertebr. Pathol. 57: 227-233) compared virus production in two uncloned lines and in cloned lines obtained from the parent lines. There was no difference in the amount of ECV produced in the two uncloned lines and only about a two-fold difference in the OB produced. However, cloned cell lines were obtained that produced 5 times more polyhedra per ml of culture than the parent line. The number of polyhedra per infected cell was not significantly different, thus the increase in yield/ml apparently was achieved by eliminating non-producing cells. The amount of ECV expressed as plaque forming units/ml was not increased in the cloned cells. Overall in this study the yield of OBs from the best cloned cell line was about 85-fold better than from the poorest producing cell line.
In addition to needing cell lines capable of producing high yields of insect viruses for use in pest control research, baculoviruses are also used to produce large amounts of recombinant, foreign proteins of medical, pharmaceutical, and veterinary importance in various insect cell lines. xe2x80x9cRecombinant, foreign proteinsxe2x80x9d refer to peptides or proteins, unrelated to, or, other than, the proteins of the wild-type baculovirus and to peptides or proteins which are not intrinsically found in the wild-type baculovirus in either the position or copy number provided in the genetically engineered baculovirus. The desired product is an expressed protein that is produced in large amounts and that is as similar to the natural protein as possible, including necessary post-translation processing and modification. Growing cells in suspension in large volumes and in medium free of fetal bovine serum (FBS) is preferred in order to facilitate downstream recovery and purification of expressed protein and to reduce costs (Vaughn, J. 1999. In: Encyclopedia of Bioprocess Technology: Fermentation, Biocatalysis, and Bioseparation, Flickinger et al., Eds. John Wiley and Sons, Inc., NY, pages 1444-1457).
Differences in yields of expressed gene products from engineered baculoviruses among cell lines have been reported. Hink et al. (1991. BiotechnoL Prog. 7: 9-14) compared the expression of three recombinant proteins in twenty-three different cell lines. For each protein, the yield varied among the cell lines and no one cell line produced the highest yields for all three proteins. However, the IZD-MB0503 would be a good compromise for producing all three proteins in a single line. A clone from this line that grew well in suspension was selected and cultured in a ten liter fermentor.
Two of the same lines, IPLB-Hv-T1 and IPLB-LdElta, also were tested by Betenbaugh et aL. (1991. Biotechnol. Prog. 7: 462-467) for their ability to produce the recombinant protein xcex2-galactosidase and a porcine rotaviral protein, VP4.In this study, the yields of xcex2-galactosidase from both cell lines exceeded that of Sf-9 in activity/ml culture. However, in terms of activity/mg of protein produced, only the yield from LdElta exceeded that of Sf-9. Yields from Sf-9 cells obtained in this study were lower than those reported by Hink et al. (supra), possibly due to differences in culture conditions. Total production of VP4 in LdElta cells was almost double that of Sf-9 cells. More of the VP4 was released from the SF-9 cells than from the LdElta cells; 95% and 88%, respectively. Both cell lines grew in suspension culture, but the LdElta cells tended to aggregate, which could present a problem in larger vessels.
Recently, a clone of an embryonic cell line T. ni developed by Granados and co-workers (Granados et al. 1994. J. Invertebr. Pathol. 64: 260-266) has been shown to produce high yields of wild type virus and several recombinant proteins. In the first 3-6 virus passages, polyhedra production was greater than 500 polyhedralcell with specific activity equal to that of polyhedra produced in larvae. As with other cell lines, both the number of polyhedra and the specific activity declined after the 6th virus passage, with production stabilizing at about 100 polyhedra per cell after the 19th passage. The cloned line, BTI-TN-5B1-4, xe2x80x9chigh fivexe2x80x9d, originally developed as a substrate dependent line, has been adapted to grow in suspension.
Insect cell lines are used as a culture system for the production of diagnostics and vaccines used in human and veterinary medicine. Hundreds of recombinant proteins have been expressed in insect cells and they are immunogenically, antigenically, and functionally similar to the native proteins. Among the post-translational processing steps that have been shown to occur in insect cells are fatty acid acylation, phosphorylation, and-glycosylation (Luckow, V. A. 1995. In: Baculoviruses Expression Systems and Biopesticides, Shuler et al., Eds. Wiley-Liss, New York, N.Y. , pages 51-90). The full nature of the glycosylation of proteins in insect cells is unclear. Most of the proteins recovered from insect cell cultures migrated faster on SDS-PAGE gels than the native protein, indicating a lower molecular weight because of incomplete post-translational modification. N-glycosylation has been described in many baculovirus-cell systems. In question is the ability of insect cells to produce proteins with complex oligosaccharides containing sialic acid residues. The absence of these residues can alter the biological activity of the protein. Davis and Wood (1995. In Vitro Cell. and Develop. Biol. 31: 659-663) examined the glycosylation of the human placental secreted alkaline phosphatase in insects and in the insect cell lines Sf-21, TN-368, and BTI-Tn-5B1-4. In the insect larvae, the protein contained complex oligosaccharide having sialic acid, but, that produced in the cell lines had only simple-type glycans. Davidson and Casttellino (1991. Biochemistry 30: 6689-6696) reported that in the human plasminogen produced in the cell line IZD-MB0503, M. brassicae, 63% of the oligosaccharides were of the complex type. These findings indicate that if the recombinant protein requires the complex oligosaccharide, additional cell lines should be tested. Other post-translational modifications may also require choosing a cell line other than one of those commonly used.
Thus, insect cell lines can be an economical means for obtaining high yields of bacculovirus and large quantities of recombinant proteins. Unlike many mammalian cells used for this purpose, they grow readily in suspension, and do not require a special atmosphere. Therefore, they can be grown in large stirred tanks with minimal specialized equipment. They infect only arthropods and therefore pose no risk to mammals. Yields of protein from baculovirus expression vectors in insect cell cultures are 20-250 times higher than those from mammalian cells. There are a number of protein-free media available from commercial supplies and the yields of foreign gene products from cells grown in these media generally have been comparable to yields in FBS-supplemented media. The baculovirus-insect cell system has been used with over five hundred genes; ninety-five percent of the expressed proteins have been biologically active (Vaughn, J., supra). However, although many cell lines have been described in the art, the need exists for new cell lines as a means for producing high yields of virus and for expressing and obtaining large quantities of a wide range of functional proteins.
Accordingly, the present invention provides cell lines which can be used to generate large quantities of genetically engineered baculovirus and large quantities of functional expressed protein.
We have now discovered novel cell lines which are useful for the production of large quantities of viral agents (viruses, viral particles, and/or occlusion bodies) or mixtures thereof in vitro.
In accordance with this discovery, it is an object of the invention to provide novel cell lines and a method of using them to produce high yields of viral agents, particularly baculoviruses. The viral agents may be employed as biological control agents.
It is also an object of the invention to provide cell lines which are effective for the production of functional expressed protein.
It is a further object of the invention to provide a method of using the cell lines for the production of genetically engineered baculovirus which express foreign proteins of medical, pharmaceutical, and veterinary importance.
Another object of the invention is to provide cell lines which are tolerant of a wide range of recombinant products.
Other objects and advantages of this invention will become readily apparent from the ensuing description.